Maria E. Ariza

Lead and mercury mutagenesis: Role of H2O2, superoxide dismutase, and xanthine oxidase

It has been suggested that reactive oxygen intermediates (ROIs) may have a role in the genotoxic effects of lead (Pb2+) and mercury (Hg2+), but there have not been any definitive studies demonstrating a causal relationship between the induction of ROIs by these metals and mutagenesis. We previously demonstrated, using the transgenic Chinese hamster ovary cell line AS52, that low concentrations (0.1–1 μM) of Pb2+ and Hg2+ are mutagenic. In the present study, using a novel histochemical computer‐enhanced image analysis technique, we demonstrate that Pb2+ and Hg2+ induce the formation of H2O2 in AS52 cells by at least two distinct mechanisms. One is characterized by the rapid induction of H2O2 following treatment of cells with concentrations of Pb2+ or Hg2+ below 0.8 and 1 μM, respectively, while the second occurs in AS52 cells treated with concentrations of Pb2+ or Hg2+ greater than 0.8 and 1 μM, respectively. Pb2+ and Hg2+ (0.1–1 μM) had no effect on the activities of partially purified catalase, glutathione peroxidase, or glutathione reductase, important enzymes involved with antioxidant defense, but these metals stimulated the activities of copper‐zinc superoxide dismutase (CuZn‐SOD) and xanthine oxidase (XO). Allopurinol (50 μM), a specific inhibitor of xanthine oxidase, inhibited the induction of H2O2 by Pb2+ (0.8–1 μM) and Hg2+ (1 μM) and also inhibited Pb2+‐ and Hg2+‐induced mutagenesis. These results demonstrate that Pb2+ and Hg2+ disrupt the redox status of AS52 cells by enhancing the activities of CuZn‐SOD and XO. Furthermore, the results of these studies also demonstrate that there is a causal relationship between the induction of H2O2 by these metals and mutagenesis. Environ. Mol. Mutagen. 31:352–361, 1998.

Antimutagenic and promutagenic activity of ascorbic acid during oxidative stress

Ascorbic acid (AA) has both antioxidant and prooxidant activities. However, there have not been any studies to elucidate the molecular mechanisms that exposed determine whether AA functions as an anti‐ or a prooxidant during oxidative stress. The results of this study, using the Chinese hamster ovary cell line AS52 as a model system, demonstrate that there is a temporal relationship between the anti‐ and prooxidant activities of a physiologically relevant concentration of AA (50 μM) and oxidative stress. Treatment of cells with AA (50 μM) 24 hr prior to treatment of the cells with a radical generating system (RGS) results in a statistically significant inhibition of the cytotoxicity and mutagenicity associated with exposure of AS52 cells to oxidative stress. Conversely, cotreatment of cells with AA and the RGS results in a statistically significant increase in both the cytotoxic and mutagenic effects of oxidative stress when compared to cell populations only to the RGS. The results, using a novel histo‐chemical‐computer image analysis system to detect hydrogen peroxide (H2O2), also demonstrate that there is a direct correlation between the ability of AA to decrease the levels of H2O2 in cells and the cytotoxic and mutagenic effects of oxidative stress. This study suggests that the time at which AA is administered in relation to exposure to oxidative stress has an impact on AA antimutagenic activity, and this may explain the conflicting results concerning the effectiveness of AA as a cancer chemopreventive agent. Environ. Mol. Mutagen. 30:339–345, 1997.

Epstein-Barr virus encoded dUTPase containing exosomes modulate innate and adaptive immune responses in human dendritic cells and peripheral blood mononuclear cells.

We have recently demonstrated that Epstein-Barr virus (EBV)-encoded deoxyuridine triphosphate nucleotidohydrolase (dUTPase) modulates innate immunity in human primary monocyte-derived macrophages through toll-like receptor (TLR) 2 leading to NF-κB activation and the production of pro-inflammatory cytokines. Our previous depletion studies indicated that dendritic cells (DCs) may also be a target of the EBV-encoded dUTPase. However, the role of EBV-encoded dUTPase in DC activation/function and its potential contribution to the inflammatory cellular milieu characteristic of EBV-associated diseases remains poorly understood. In the present study, we demonstrate that EBV-encoded dUTPase significantly altered the expression of genes involved in oncogenesis, inflammation and viral defense mechanisms in human primary DCs by microarray analysis. Proteome array studies revealed that EBV-encoded dUTPase modulates DC immune responses by inducing the secretion of pro-inflammatory TH1/TH17 cytokines. More importantly, we demonstrate that EBV-encoded dUTPase is secreted in exosomes from chemically induced Raji cells at sufficient levels to induce NF-κB activation and cytokine secretion in primary DCs and peripheral blood mononuclear cells (PBMCs). Interestingly, the production of pro-inflammatory cytokines in DCs and PBMCs was TLR2-dependent. Together these findings suggest that the EBV-encoded dUTPase may act as an intercellular signaling molecule capable of modulating the cellular microenvironment and thus, it may be important in the pathophysiology of EBV related diseases.

Lead and mercury mutagenesis: type of mutation dependent upon metal concentration.

Lead and mercury are toxic metals that are widely distributed in the atmosphere, soil, and groundwater. It is estimated that 2–4 × 104 tons of these metals are released annually into the environment by natural and industrial processes. Therefore, human exposure to low relatively nontoxic concentrations of these metals is unavoidable. However, the possible health effects of such exposure remain controversial. We have previously reported that low, subthreshold concentrations (0.1–1 μM) of these metals are mutagenic in the transgenic Chinese hamster ovary cell line AS52. The purpose of the present study is to determine the types of mutations induced in the gpt gene in AS52 cells. Using multiplex polymerase chain reaction and southern blot analyses, we characterized the 138 lead‐induced, 192 mercury‐induced, 29 reactive oxygen radical‐induced, and 20 spontaneously arising mutants for point and deletion mutations in the gpt gene. Similar levels of point mutations were observed in the lead‐ and mercury‐induced populations (47.8 and 53.6, respectively), which was significantly less than that occurring in the spontaneously arising and reactive oxygen intermediate‐induced mutants. However, further examination of the data revealed that at concentrations of the metals of equal to or less than 0.4 μM, the majority of the mutations in the gpt gene were point mutations, while at higher concentrations, deletions (partial and complete) were the predominant type of mutation. These results are consistent with the hypothesis that lead and mercury induce mutations in eukaryotic cells by at least two distinct mechanisms.

Mutagenesis of AS52 cells by low concentrations of lead(II) and mercury(II)

Little is known at the molecular level concerning the genotoxic effects following the acute exposure of eukaryotic cells to low concentrations of lead (II) or mercury (II). There have been conflicting reports concerning the mutagenic potential of these heavy metals, and there have not been any studies performed to determine the molecular mechanism(s) by which these metals are mutagenic. The Chinese hamster ovary cell line, AS52, contains a stably integrated single functional copy of the Escherichia coli xanthine‐guanine phosphoribosyltransferase (gpt) gene. Mutations in the gpt gene confer resistance to 6‐thioguanine (TG). There was little effect on viability, as measured by relative cloning efficiency, of AS52 cells exposed to lead (II) or mercury (II) up to concentrations of 0.5; mgrM and 0.3; mgrM, respectively. However, higher concentrations of the metals caused a significant increase in cell death. There was also a dose‐dependent increase in the isolation of mutants resistant to TG in treated cells when compared to nontreated controls. Concentrations of the metals as low as 0.1 μM caused a significant increase in the number of mutants resistant to TG when compared to the number of spontaneous mutants obtained in nontreated controls. While the molecular mechanism(s) by which lead and mercury (II) are genotoxic is unknown, the results of this study demonstrate that low concentrations of lead (II) and mercury (II) are mutagenic in eukaryotic cells.

Antibody to Epstein-Barr Virus Deoxyuridine Triphosphate Nucleotidohydrolase and Deoxyribonucleotide Polymerase in a Chronic Fatigue Syndrome Subset

Background A defined diagnostic panel differentiated patients who had been diagnosed with chronic fatigue syndrome (CFS), based upon Fukuda/Carruthers criteria. This diagnostic panel identified an Epstein-Barr virus (EBV) subset of patients (6), excluding for the first time other similar “clinical” conditions such as cytomegalovirus (CMV), human herpesvirus 6 (HHV6), babesiosis, ehrlichiosis, borreliosis, Mycoplasma pneumoniae, Chlamydia pneumoniae, and adult rheumatic fever, which may be mistakenly called CFS. CFS patients were treated with valacyclovir (14.3 mg/kg q6h) for ≥12 months. Each patient improved, based upon the Functional Activity Appraisal: Energy Index Score Healthcare Worker Assessment (EIPS), which is a validated (FSS-9), item scale with high degree of internal consistency measured by Cronbachs alpha. Methods Antibody to EBV viral capsid antigen (VCA) IgM, EBV Diffuse Early Antigen EA(D), and neutralizing antibodies against EBV-encoded DNA polymerase and EBV-encoded dUTPase were assayed serially approximately every three months for 13–16 months from sera obtained from patients with CFS (6) and from sera obtained from twenty patients who had no history of CFS. Results Antibodies to EBV EA(D) and neutralizing antibodies against the encoded-proteins EBV DNA polymerase and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) were present in the EBV subset CFS patients. Of the sera samples obtained from patients with CFS 93.9% were positive for EA(D), while 31.6% of the control patients were positive for EBV EA(D). Serum samples were positive for neutralizing antibodies against the EBV-encoded dUTPase (23/52; 44.2%) and DNA polymerase (41/52; 78.8%) in EBV subset CFS patients, but negative in sera of controls. Conclusions There is prolonged elevated antibody level against the encoded proteins EBV dUTPase and EBV DNA polymerase in a subset of CFS patients, suggesting that this antibody panel could be used to identify these patients, if these preliminary findings are corroborated by studies with a larger number of EBV subset CFS patients.

Human herpesviruses-encoded dUTPases: a family of proteins that modulate dendritic cell function and innate immunity

We have previously shown that Epstein-Barr virus (EBV)-encoded dUTPase can modulate innate immune responses through the activation of TLR2 and NF-κB signaling. However, whether this novel immune function of the dUTPase is specific for EBV or a common property of the Herpesviridae family is not known. In this study, we demonstrate that the purified viral dUTPases encoded by herpes simplex virus type 2 (HSV-2), human herpesvirus-6A (HHV-6A), human herpesvirus-8 (HHV-8) and varicella-zoster virus (VZV) differentially activate NF-κB through ligation of TLR2/TLR1 heterodimers. Furthermore, activation of NF-κB by the viral dUTPases was inhibited by anti-TLR2 blocking antibodies (Abs) and the over-expression of dominant-negative constructs of TLR2, lacking the TIR domain, and MyD88 in human embryonic kidney 293 cells expressing TLR2/TLR1. In addition, treatment of human dendritic cells and PBMCs with the herpesviruses-encoded dUTPases from HSV-2, HHV-6A, HHV-8, and VZV resulted in the secretion of the inflammatory cytokines IL-1β, IL-6, IL-8, IL-12, TNF-α, IL-10, and IFN-γ. Interestingly, blocking experiments revealed that the anti-TLR2 Ab significantly reduced the secretion of cytokines by the various herpesviruses-encoded dUTPases (p < 0.05). To our knowledge, this is the first report demonstrating that a non-structural protein encoded by herpesviruses HHV-6A, HHV-8, VZV and to a lesser extent HSV-2 is a pathogen-associated molecular pattern. Our results reveal a novel function of the virus-encoded dUTPases, which may be important to the pathophysiology of diseases caused by these viruses. More importantly, this study demonstrates that the immunomodulatory functions of dUTPases are a common property of the Herpesviridae family and thus, the dUTPase could be a potential target for the development of novel therapeutic agents against infections caused by these herpesviruses.

Evidence for the Role of Epstein Barr Virus Infections in the Pathogenesis of Acute Coronary Events

Background The role of viral infections in the pathogenesis of atherosclerosis remains controversial largely due to inconsistent detection of the virus in atherosclerotic lesions. However, viral infections elicit a pro-inflammatory cascade known to be atherogenic and to precipitate acute ischemic events. We have published in vitro data that provide the foundation for a mechanism that reconciles these conflicting observations. To determine the relation between an early viral protein, deoxyuridine triphosphate nucleotidohydrolase (dUTPase), produced following reactivation of Epstein Barr Virus (EBV) to circulating pro-inflammatory cytokines, intercellular adhesion molecule-1 (ICAM-1) and acute coronary events. Methodology/Principal Findings Blood samples were obtained from 299 patients undergoing percutaneous coronary intervention for stable angina (SA), unstable angina (UA), or acute myocardial infarction (AMI). Plasma concentrations of pro-inflammatory cytokines and neutralizing antibody against EBV-encoded dUTPase were compared in the three patient groups. AMI was associated with the highest measures of interleukin-6 (ANOVA p<0.05; 4.6±2.6 pg/mL in patients with AMI vs. 3.2±2.3 pg/mL in SA). ICAM-1 was significantly higher in patients with AMI (ANOVA p<0.05; 304±116 pg/mL in AMI vs. 265±86 pg/mL SA). The highest values of ICAM-1 were found in patients having an AMI and who were antibody positive for dUTPase (ANOVA p = 0.008; 369±183 pg/mL in AMI and positive for dUTPase vs. 249±70 pg/mL in SA negative for dUTPase antibody). Conclusions/Significance These clinical data support a model, based on in vitro studies, by which EBV may precipitate AMI even under conditions of low viral load through the pro-inflammatory action of the early protein dUTPase that is produced even during incomplete viral replication. They further support the putative role of viral infections in the pathogenesis of atherosclerosis and coronary artery events.

Myalgic encephalomyelitis/chronic fatigue syndrome and gulf war illness patients exhibit increased humoral responses to the herpesviruses‐encoded dUTPase: Implications in disease pathophysiology

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Gulf War Illness (GWI) are debilitating diseases with overlapping symptomology and there are currently no validated tests for definitive diagnosis of either syndrome. While there is evidence supporting the premise that some herpesviruses may act as possible triggers of ME/CFS, the involvement of herpesviruses in the pathophysiology of GWI has not been studied in spite of a higher prevalence of ME/CFS in these patients. We have previously demonstrated that the deoxyuridine triphosphate nucleotidohydrolases (dUTPase) encoded by Epstein‐Barr virus (EBV), human herpesvirus‐6 (HHV‐6), and varicella‐zoster virus (VZV) possess novel functions in innate and adaptive immunity. The results of this study demonstrate that a significant percentage of patients with ME/CFS (30.91‐52.7%) and GWI (29.34%) are simultaneously producing antibodies against multiple human herpesviruses‐encoded dUTPases and/or the human dUTPase when compared to controls (17.21%). GWI patients exhibited significantly higher levels of antibodies to the HHV‐6 and human dUTPases than controls (P = 0.0053 and P = 0.0036, respectively), while the ME/CFS cohort had higher anti‐EBV‐dUTPase antibodies than in both GWI patients (P = 0.0008) and controls (P < 0.0001) as well as significantly higher anti‐human dUTPase antibodies than in controls (P = 0.0241). These results suggest that screening of patients’ sera for the presence of various combinations of anti‐dUTPase antibodies could be used as potential biomarkers to help identify/distinguish patients with these syndromes and better direct treatment.

Mutagenic potential of peripheral blood leukocytes : in vivo exposure to the carcinogen 7,12-dimethylbenz [a] anthracene, and the tumor promoter 12-O-tetradecanoylphorbol acetate followed by in vitro co-culture with AS52 cells

Co-culture of AS52 cells with peripheral blood leukocytes (PBLs), obtained from SENCAR mice topically treated with either tetradecanoyl-phorbol-13-acetate (TPA) or 7,12-dimethylbenz[a]anthracene (DMBA)-TPA, resulted in a 7-160-fold increase in the mutation frequency of the gpt gene in AS52 cells when compared to that induced by PBLs isolated from mice treated with either acetone or DMBA. This increase in mutation frequency was inhibited by the anti-oxidant (-)epigallocatechin gallate (EGCG). These results demonstrate that the AS52 cell line can be used as a mammalian mutagenesis model for the study of in vivo mechanism(s) of mutagenesis by leukocytes and also as a model for in vivo chemoprevention studies.